Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans. Variola causes smallpox, which has a 30-40% mortality. Protocols for vaccination rely on the vaccinia virus which has multiple, potential side effects. The United States stopped vaccinating the public in 1972, while worldwide vaccination terminated in the late 1970s. Only 10-20% of individuals previously vaccinated are still protected due to waning immunity. Public health concerns regarding the re-emergence of variola virus has led to renewed interest in the pathogenesis of smallpox. Since ethical and public health concerns preclude in vivo work on variola virus, and the World Health Organization prohibits DNA recombination studies between variola and other Orthopoxvirus genomes, studies of variola require circuitous approaches. In vivo model systems involving orthopoxviruses do exist but are limited primarily to vaccinia, cowpox, and ectromelia viruses, which do not cause disease in immunocompetent humans (Moss (1996) Proc. Natl. Acad. Sci. USA 93:11341-11348). Therefore, the relative benignity of other orthopoxviruses in humans may underestimate the importance of any homologous protein in the pathogenesis of smallpox.
Protocols for resuming the administration of smallpox virus also have serious implications. In the last decades, the growing number of immunocompromised patients suffering from AIDS, diabetes, cancer and other conditions has increased. In addition, the widespread use of immunosuppressants for organ transplant patients, the common practice of radiation and chemotherapy for treating malignancies, as well as the growing size of the aging population have also increased. Administration of the current smallpox vaccine and subsequent shedding of this virus may result in widespread vaccinia virus infection in the population of immunocompromised individuals. Therefore, alternative smallpox vaccines are imperative at this time.
Complement regulatory proteins (CRPs), encoded by genes located in the terminal regions of orthopoxviruses, are important for viruses to evade a host-mediated complement attack (Cooper, N. R. Complement and Viruses. In: The Human complement System in Health and Disease, Vol. 1 (eds. Volanakis, J. E. and Frank, M. M.) 393-407, Marcel Dekker, Inc. NY, 1998). Virally-encoded CRPs presumably deflect complement destruction of infected host cells to allow for more efficient viral spread (Shchelkunov et al. (1993) FEBS Lett. 319:80-83; Lachmann and Davies (1997) Immunological Reviews 159:69-77). CRPs differ with respect to ligand specificity (C3b and/or C4b) and the mechanism of convertase inactivation. They may accelerate the normal decay of the classical and alternative pathway convertases or function as cofactors for the serine protease, factor I, to enzymatically cleave the xcex1xe2x80x2 chains of C3b and C4b into smaller, inactive fragments (Liszewski and Atkinson. Regulatory Proteins of Complement. In: The Human complement System in Health and Disease, Vol. 1 (eds. Volanakis, J. E. and Frank, M. M.) 149-166, Marcel Dekker, Inc. NY, 1998). Structurally, CRPs are composed of 4-56 homologous motifs, termed short consensus repeats (SCR).
Vaccinia Virus Complement Control Protein (VCP) is a CRP produced by vaccinia virus that has been shown to enhance the virulence of vaccinia in rabbit and guinea pig experimental models and causes larger lesions when injected intradermally (Isaacs et al. (1992) Proc. Natl. Acad. Sci. USA 89:628-632). VCP functions primarily as a cofactor for factor I rather than as a decay accelerator (Kotwal et al. (1990) Science 250:827-830; McKenzie et al. (1992) J. Infect. Dis. 166:1245-1250; Sahu et al. (1998) J. Immunol. 160:5596-5604). Hence, Kotwal et al. (U.S. Pat. No. 5,157,110) describe the use of VCP to bind to C4b and inhibit the complement cascade. Furthermore, Rosengard et al. (U.S. Pat. No. 5,843,778) disclose the use of recombinant, VCP-immunoglobulin Fc region fusion protein to modulate complement activation through binding of complement components C3b and C4b.
DNA comparison studies revealed that the genomes of all variola virus strains also encode a CRP homolog consisting of 4 SCRs (Massung et al. (1996) Virology 221:291-300). This Smallpox Inhibitor of Complement Enzyme (SPICE) differs from the VCP amino acid sequence by 4.6%; the 11 amino acid differences are dispersed throughout SCR2, 3, and 4. In the absence of wild-type variola proteins, Rosengard et al. (PCT Publication WO 99/44625) describes a SPICE polypeptide that has been generated by molecular engineering of VCP. The intended use of this SPICE protein, fused to an immunoglobulin molecule, is to modulate complement activation.
To date, there are few options for the rapid detection of smallpox infection. Ropp et al. ((1995) J. Clin. Microbiol. 33:2069-76) demonstrate the use of a combined PCR amplification-endonuclease digestion of the hemagglutinin gene to identify and differentiate smallpox from other orthopoxviruses. Moreover, Hooper et al. (U.S. patent application Ser. No. 20020009447, filed Jan. 24, 2002) describe the use of monoclonal antibodies directed to vaccinia L1R and A33R antigens to detect, prevent, and/or treat vaccinia virus infections in vitro and in vivo. Monoclonal antibodies directed to orthopoxvirus homologs of vaccinia L1R and A33R are also disclosed.
The present invention addresses the needs of improved detection, prevention, and treatment of variola virus infection. Antibodies directed against SPICE protein and SPICE-specific primers for PCR amplification of the SPICE gene are provided for the detection of variola virus. The present invention also addresses the need for improved vaccines for the prevention of variola virus infection by providing antibodies directed against SPICE for passive vaccination. Furthermore, the present invention provides SPICE fusion proteins for vaccination against smallpox virus. Treatment for variola virus infections is provided through administration of an antibody directed against SPICE. Finally, SPICE fusion proteins are described for binding human complement components to modulate complement activation.
The present invention provides antibodies against Smallpox Inhibitor of Complement Enzyme (SPICE).
An object of the present invention is to provide a method of preventing or treating variola virus infection by administering anti-SPICE antibodies.
Another object of the present invention is to provide a method of co-administering anti-VCP/SPICE antibodies with vaccinia virus vaccine to modulate vaccine side effects.
Another object of the present invention is to provide a method for detecting variola virus infections in humans by detecting the presence of SPICE protein or nucleic acid sequences encoding SPICE.
A further object of the present invention provides kits for the detection of SPICE or nucleic acid sequences encoding SPICE.
Another object of the present invention is to provide a vaccine for variola virus by administering recombinant SPICE and VCP proteins to enhance the immune response to variola virus or administering a SPICE or VCP DNA vaccine.
A further object of the present invention is directed to the modulation of complement activation by administering recombinant SPICE and VCP proteins.
The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth below.